Construction and identification of miR-195 silencing transgenic vector with temporal and spatial regulation

Objective The purpose of this study is to construct a validated miR-195 silencing transgenic vector with temporal and spatial regulation. Methods Five different regulatory elements (a neuron-specific promoter, Tet-on tetracycline inducible expression system, miR-195 sponge, green fluorescent protein and cHs4 insulators) separately were used to construct the transgenic vector by molecular cloning techniques. After a sequencing confirmation, the newly established vector was transiently transfected in SH-SY5Y cells; the transfection efficiency was confirmed by fluorescence microscopy; Western blot showed the GFP protein expression. MiR-195 expression level was measured by real-time quantitative PCR. Results The reverse Tet Transactivator (rtTA) element of the Tet-on advanced system was cloned into the downstream of the NSE promoter, and the fragment of the miR-195 sponge that carrying IRES-mediated EGFP was constructured into the multiple cloning site of the TRE-Tight vector. The regulatory element (NSE-rtTA) and the response element (TRE-miR-195 sponge-IRES-EGFP) were introduced in a pcDNA3.1+ backbone with intervals by three different regional 2× cHs4 insulator core fragments to construct a neuron-specific miR-195 silencing transgenic vector. The vector was transiently cotransfected with miR-195 in the SH-SY5Y cells treated with Doxycycline inducers. The green fluorescence was visible in the cytoplasm of the treated cells and the GFP proteins can be detected, and the miR-195 level was also significantly reduced compare with the miR-195 transfected cells. Conclusion Neuron-specific miR-195 silencing transgenic vector was successfully constructed. The vector exhibited concurrent temporal and spatial regulation characteristic in vitro. The successful construction will lay foundations for the establishment of transgenic mouse models. Key words: miR-195; Transgenic vector; Tet-on tetracycline inducible expression system